Nematicidal activity of paucimannose-type glycoconjugates from acacia honey.

Natural honey contains glycoconjugates as minor components. We characterized acacia honey glycoconjugates with molecular masses in the range of 2-5 kDa. The glycoconjugates were separated by RP-HPLC into three peaks (termed RP-2-5 k-I, RP-2-5 k-II, and RP-2-5 k-III) which demonstrated paralyzing effects on the model nematode C. elegans (ED50 of 50 ng glycoconjugates/µL). To examine molecular mechanisms underlying the nematicidal effects of honey glycoconjugates, expressional analyses of genes that are essential for the growth, development, reproduction, and movement of C. elegans were carried out. Quantitative PCR-based assays showed that these molecules moderately regulate the expression of genes involved in the citric acid cycle (mdh-1 and idhg-1) and cytoskeleton (act-1 and act-2). MALDI-ToF-MS/MS analysis of RP-HPLC peaks revealed the presence of paucimannose-like N-glycans which are known to play important roles in invertebrates e.g., worms and flies. These findings provided novel information regarding the structure and nematicidal function of honey glycoconjugates.

Structural insights into a cooperative switch between one and two FimH bacterial adhesins binding pauci- and high-mannose type N-glycan receptors.

The FimH type-1 fimbrial adhesin allows pathogenic Escherichia coli to adhere to glycoproteins in the epithelial linings of human bladder and intestinal tract, by using multiple fimbriae simultaneously. Pauci- and high-mannose type N-glycans are natural FimH receptors on those glycoproteins. Oligomannose-3 and oligomannose-5 bind with the highest affinity to FimH by using the same Manα1,3Man branch. Oligomannose-6 is generated from oligomannose-5 in the next step of the biogenesis of high-mannose N-glycans, by the transfer of a mannose in α1,2-linkage onto this branch. Using serial crystallography and by measuring the kinetics of binding, we demonstrate that shielding the high-affinity epitope drives the binding of multiple FimH molecules. First, we profiled FimH glycan binding on a microarray containing paucimannosidic N-glycans and in a FimH LEctPROFILE assay. To make the transition to oligomannose-6, we measured the kinetics of FimH binding using paucimannosidic N-glycans, glycoproteins and all four α-dimannosides conjugated to bovine serum albumin. Equimolar mixed interfaces of the dimannosides present in oligomannose-6 and molecular dynamics simulations suggest a positive cooperativity in the bivalent binding of Manα1,3Manα1 and Manα1,6Manα1 dimannosides. The binding of core α1,6-fucosylated oligomannose-3 in cocrystals of FimH is monovalent but interestingly the GlcNAc1-Fuc moiety retains highly flexibility. In cocrystals with oligomannose-6, two FimH bacterial adhesins bind the Manα1,3Manα1 and Manα1,6Manα1 endings of the second trimannose core (A-4'-B). This cooperative switch towards bivalent binding appears sustainable beyond a molar excess of oligomannose-6. Our findings provide important novel structural insights for the design of multivalent FimH antagonists that bind with positive cooperativity.

Evaluation of the context of downstream N- and free N-glycomic alterations induced by swainsonine in HepG2 cells.

Swainsonine (SWA), a potent inhibitor of class II α-mannosidases, is present in a number of plant species worldwide and causes severe toxicosis in livestock grazing these plants. The mechanisms underlying SWA-induced animal poisoning are not fully understood. In this study, we analyzed the alterations that occur in N- and free N-glycomic upon addition of SWA to HepG2 cells to understand better SWA-induced glycomic alterations. After SWA addition, we observed the appearance of SWA-specific glycomic alterations, such as unique fucosylated hybrid-type and fucosylated M5 (M5F) N-glycans, and a remarkable increase in all classes of Gn1 FNGs. Further analysis of the context of these glycomic alterations showed that (fucosylated) hybrid type N-glycans were not the precursors of these Gn1 FNGs and vice versa. Time course analysis revealed the dynamic nature of glycomic alterations upon exposure of SWA and suggested that accumulation of free N-glycans occurred earlier than that of hybrid-type N-glycans. Hybrid-type N-glycans, of which most were uniquely core fucosylated, tended to increase slowly over time, as was observed for M5F N-glycans. Inhibition of swainsonine-induced unique fucosylation of hybrid N-glycans and M5 by coaddition of 2-fluorofucose caused significant increases in paucimannose- and fucosylated paucimannose-type N-glycans, as well as paucimannose-type free N-glycans. The results not only revealed the gross glycomic alterations in HepG2 cells induced by swainsonine, but also provide information on the global interrelationships between glycomic alterations.

N-acetyl-ß-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical truncation pathway in human neutrophils.

We recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unusual and highly truncated paucimannosidic N-glycans (Man1-3GlcNAc2Fuc0-1), but their biosynthesis remains elusive. Guided by the well-characterized truncation pathway in invertebrates and plants in which the N-acetyl-ß-D-hexosaminidase (Hex) isoenzymes catalyze paucimannosidic protein (PMP) formation, we here set out to test if the homologous human Hex α and ß subunits encoded by HEXA and HEXB drive a similar truncation pathway in human neutrophils. To this end, we performed quantitative glycomics and glycoproteomics of several CRISPR-Cas9-edited Hex-disrupted neutrophil-like HL-60 mutants (HEXA-KO and HEXB-KO) and matching unedited cell lines. Hex disruption was validated using next-generation sequencing, enzyme-linked immunosorbent assay (ELISA), quantitative proteomics and Hex activity assays. Excitingly, all Hex-disrupted mutants displayed significantly reduced levels of paucimannosylation, particularly Man2-3GlcNAc2Fuc1, relative to unedited HL-60 suggesting that both HEXA and HEXB contribute to PMP formation via a hitherto unexplored truncation pathway in neutrophils. Quantitative N-glycomics indeed demonstrated reduced utilization of a putative noncanonical truncation pathway in favor of the canonical elongation pathway in all Hex-disrupted mutants relative to unedited controls. Quantitative glycoproteomics recapitulated the truncation-to-elongation switch in all Hex-disrupted mutants and showed a greater switch for N-glycoproteins cotrafficking with Hex to the azurophilic granules of neutrophils such as myeloperoxidase. Finally, we supported the Hex-PMP relationship by documenting that primary neutrophils isolated from an early-onset Sandhoff disease patient (HEXB-/-) displayed dramatically reduced paucimannosylation relative to neutrophils from an age-matched unaffected donor. We conclude that both human Hex α and ß mediate PMP formation via a putative noncanonical truncation pathway in neutrophils.

Production of Human Acid-Alpha Glucosidase With a Paucimannose Structure by Glycoengineered Arabidopsis Cell Culture.

Plant cell cultures have emerged as a promising platform for the production of biopharmaceutics due to their cost-effectiveness, safety, ability to control the cultivation, and secrete products into culture medium. However, the use of this platform is hindered by the generation of plant-specific N-glycans, the inability to produce essential N-glycans for cellular delivery of biopharmaceutics, and low productivity. In this study, an alternative acid-alpha glucosidase (GAA) for enzyme replacement therapy of Pompe disease was produced in a glycoengineered Arabidopsis alg3 cell culture. The N-glycan composition of the GAA consisted of a predominantly paucimannosidic structure, Man3GlcNAc2 (M3), without the plant-specific N-glycans. Supplementing the culture medium with NaCl to a final concentration of 50 mM successfully increased GAA production by 3.8-fold. GAA from an NaCl-supplemented culture showed a similar N-glycan profile, indicating that the NaCl supplementation did not affect N-glycosylation. The results of this study highlight the feasibility of using a glycoengineered plant cell culture to produce recombinant proteins for which M3 or mannose receptor-mediated delivery is desired.

Tumor cells express pauci- and oligomannosidic N-glycans in glycoproteins recognized by the mannose receptor (CD206).

The macrophage mannose receptor (CD206, MR) is an endocytic lectin receptor which plays an important role in homeostasis and innate immunity, however, the endogenous glycan and glycoprotein ligands recognized by its C-type lectin domains (CTLD) have not been well studied. Here we used the murine MR CTLD4-7 coupled to the Fc-portion of human IgG (MR-Fc) to investigate the MR glycan and glycoprotein recognition. We probed 16 different cancer and control tissues using the MR-Fc, and observed cell- and tissue-specific binding with varying intensity. All cancer tissues and several control tissues exhibited MR-Fc ligands, intracellular and/or surface-located. We further confirmed the presence of ligands on the surface of cancer cells by flow cytometry. To characterize the fine specificity of the MR for glycans, we screened a panel of glycan microarrays. Remarkably, the results indicate that the CTLD4-7 of the MR is highly selective for specific types of pauci- and oligomannose N-glycans among hundreds of glycans tested. As lung cancer tissue and the lung cancer cell line A549 showed intense MR-Fc binding, we further investigated the MR glycoprotein ligands in those cells by immunoprecipitation and glycoproteomic analysis. All enriched glycoproteins, of which 42 were identified, contained pauci- or oligomannose N-glycans, confirming the microarray results. Our study demonstrates that the MR CTLD4-7 is highly selective for pauci- and oligomannosidic N-glycans, structures that are often elevated in tumor cells, and suggest a potential role for the MR in tumor biology.

Analysis of N-glycan profile of Arabidopsis alg3 cell culture.

N-Glycosylation is essential for protein stability, activity and characteristics, and is often needed to deliver pharmaceutical glycoproteins to target cells. A paucimannosidic structure, Man3GlcNAc2 (M3), has been reported to enable cellular uptake of glycoproteins through the mannose receptor (MR) in humans, and such uptake has been exploited for the treatment of certain diseases. However, M3 is generally produced at a very low level in plants. In this study, a cell culture was established from an Arabidopsis alg3 mutant plant lacking asparagine-linked glycosylation 3 (ALG3) enzyme activity. Arabidopsis alg3 cell culture produced glycoproteins with predominantly M3 and GlcNAc-terminal structures, while the amount of plant-specific N-glycans was very low. Pharmaceutical glycoproteins with these characteristics would be valuable for cellular delivery through the MR, and safe for human therapy.

Neutrophil myeloperoxidase harbors distinct site-specific peculiarities in its glycosylation.

Anti-neutrophil cytoplasmic autoantibodies (ANCAs) are directed against lysosomal components of neutrophils. ANCAs directed to proteinase 3 and myeloperoxidase (MPO) in particular are associated with distinct forms of small vessel vasculitides. MPO is an abundant neutrophil-derived heme protein that is part of the antimicrobial defense system. The protein is typically present in the azurophilic granules of neutrophils, but a large portion may also enter the extracellular space. It remains unclear why MPO is frequently the target of antibody-mediated autoimmune responses. MPO is a homodimeric glycoprotein, posttranslationally modified with complex sugars at specific sites. Glycosylation can strongly influence protein function, affecting its folding, receptor interaction, and backbone accessibility. MPO potentially can be heavily modified as it harbors 5 putative N-glycosylation sites (10 in the mature dimer). Although considered important for MPO structure and function, the full scope and relative abundance of the glycans attached to MPO is unknown. Here, combining bottom-up glycoproteomics and native MS approaches, we structurally characterized MPO from neutrophils of healthy human donors. We quantified the relative occupancy levels of the glycans at each of the five sites and observed complex heterogeneity and site-specific glycosylation. In particular, we detected glycosylation phenotypes uncommon for glycoproteins in the extracellular space, such as a high abundance of phosphorylated high-mannose species and severely truncated small glycans having the size of paucimannose or smaller. We hypothesize that the atypical glycosylation pattern found on MPO might contribute to its specific processing and presentation as a self-antigen by antigen-presenting cells.

Diversity and functions of protein glycosylation in insects.

The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes.

Site specific N-glycan profiling of NeuAc(α2-6)-Gal/GalNAc-binding bark Sambucus nigra agglutinin using LC-MSn revealed differential glycosylation.

The bark of Sambucus nigra contains a complex mixture of glycoproteins that are characterized as chimeric lectins known as type II ribosome inactivating proteins and holo lectins. These type II ribosome inactivating proteins possess RNA N-glycosidase activity in subunit A and lectin activity associated with subunit B exhibiting distinct sugar specificities to NeuAc(α2-6)-Gal/GalNAc and Gal/GalNAc. In the present study we have determined the N-glycosylation pattern of type II ribosome inactivating protein specific to NeuAc(α2-6)-Gal/GalNAc (Sambucus nigra agglutinin I) by subjecting it to digestion with multiple proteases. The resulting mixture of peptides and N-glycopeptides were analyzed on liquid chromatography coupled to electro spray ionization-iontrap mass spectrometry in MSn mode. MS2 of precursor ions was carried out using CID which provided information on glycan sequence. In subsequent MS3 of Y1/Y1α ions (peptide + HexNAc)+n of corresponding N-glycopeptides, resulted in the fragmentation of peptide backbone confirming the site of attachment. We observed microheterogeneity in each glycan occupied site with subunit A possessing four N-glycans out of six sites with complex and paucimannose types while subunit B comprises occupancy of two sites with a paucimannose and a high mannose type. The differential N-glycosylation of subunits in SNA is discussed in the context of other type II RIPs glycans.

Complementary LC-MS/MS-Based N-Glycan, N-Glycopeptide, and Intact N-Glycoprotein Profiling Reveals Unconventional Asn71-Glycosylation of Human Neutrophil Cathepsin G.

Neutrophil cathepsin G (nCG) is a central serine protease in the human innate immune system, but the importance of its N-glycosylation remains largely undescribed. To facilitate such investigations, we here use complementary LC-MS/MS-based N-glycan, N-glycopeptide, and intact glycoprotein profiling to accurately establish the micro- and macro-heterogeneity of nCG from healthy individuals. The fully occupied Asn71 carried unconventional N-glycosylation consisting of truncated chitobiose core (GlcNAcß: 55.2%; Fucα1,6GlcNAcß: 22.7%), paucimannosidic N-glycans (Manß1,4GlcNAcß1,4GlcNAcß: 10.6%; Manß1,4GlcNAcß1,4(Fucα1,6)GlcNAcß: 7.9%; Manα1,6Manß1,4GlcNAcß1,4GlcNAcß: 3.7%, trace level of Manα1,6Manß1,4GlcNAcß1,4(Fucα1,6)GlcNAcß), and trace levels of monoantennary α2,6- and α2,3-sialylated complex N-glycans. High-resolution/mass accuracy LC-MS profiling of intact nCG confirmed the Asn71-glycoprofile and identified two C-terminal truncation variants at Arg243 (57.8%) and Ser244 (42.2%), both displaying oxidation of solvent-accessible Met152. Asn71 appeared proximal (~19 Å) to the active site of nCG, but due to the truncated nature of Asn71-glycans (~5-17 Å) we questioned their direct modulation of the proteolytic activity of the protein. This work highlights the continued requirement of using complementary technologies to accurately profile even relatively simple glycoproteins and illustrates important challenges associated with the analysis of unconventional protein N-glycosylation. Importantly, this study now facilitates investigation of the functional role of nCG Asn71-glycosylation.

Cystic fibrosis and bacterial colonization define the sputum N-glycosylation phenotype.

Although mucin O-glycosylation of sputum from individuals suffering from cystic fibrosis (CF) is known to be altered relative to their unaffected counterparts, protein N-glycosylation of CF sputum remains structurally and functionally under-characterized. We report the first N-glycome of soluble proteins in sputum derived from five CF patients, two pathogen-free and two pathogen-infected/colonized non-CF individuals suffering from other pulmonary conditions. N-Glycans were profiled using porous graphitized carbon-liquid chromatography-negative ion-tandem mass spectrometry following enzymatic release from sputum proteins. The composition, topology and linkage isomers of 68 N-glycans were characterized and relatively quantified. Recurring structural features in all sputum N-glycomes were terminal α2,6-sialylation, α1,6-core fucosylation, ß1,4-bisecting GlcNAcylation and compositions indicating paucimannosylation. Despite covering different genotypes, age, gender and microbial flora, the sputum N-glycomes showed little interpatient and longitudinal variation within CF patients. Comparative N-glycome analysis between inter-patient group revealed that lung infection/colonization, in general, extensively enriches the CF sputum N-glycosylation phenotype with paucimannose with simultaneous over-sialylation/fucosylation and under-bisecting GlcNAcylation of complex/hybrid N-glycans. In contrast, the sputum from CF patients had only slightly increased abundance of paucimannose N-glycans relative to pathogen-infected/colonized non-CF individuals. Similar to mucin O-glycosylation, protein N-glycosylation appears to be regulated primarily as a secondary effect of bacterial infection and inflammation rather than the CF pathogenesis in sputum. This study provides new structural N-glycan information to help understand the complex cellular and molecular environment of the CF affected respiratory tract.